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Biochemical Tests:

Biochemical Tests

Objectives:

Objectives Tests to know Indole Methyl Red/ Voges Proskauer Citrate H 2 S production Urea hydrolysis Motility Mannitol fermentation Lactose fermentation Coagulase Sugar ang Gas Production.

Indole Production Test :

Indole Production Test The amino acid tryptophan can be broken down by enzyme tryptophanase to form indole , pyruvic acid and ammonia as end products. Tryptophanase differentiates indole -positive enterics , such as E. coli and P.vulgaris from indole -negative enterics , such as S. marcescens . Media and Reagents: SIM with tryptophan and Kovac’s reagent. Method: Inoculate medium and incubate at 37°C for 24-48 hours. After incubation, add five drops of Kovac’s reagent to the surface. Do not stir or shake the tube. Expected Results: Positive test : Kovac’s reagent combines with indole and turns the surface red. Negative test: No red color development

Results for indole test:

Results for indole test

Methyl Red Test:

Methyl Red Test Methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. If they use mixed acid pathway and produce acidic products, then they are called methyl-red-positive. If they use butylene glycol pathway and produce neutral end products, then they are called methyl-red-negative. Media and reagents: MR-VP medium and methyl red indicator Method: Inoculate broth and incubate at 37°C for 2-5 days. After incubation, transfer 2.5 ml of inoculate to another tube and add five drops of methyl red. Roll between the palms of hands to disperse methyl red. Expected results: Positive test: acids + methyl red = red solution Negative test: neutral end products + methyl red = yellow color

Results for Methyl Red Test :

Results for Methyl Red Test Positive Negative

Voges Proskauer Test:

Voges Proskauer Test It is used to identify enteric bacteria based on their pattern of glucose metabolism. The enterics that produce neutral end-products, such as acetoin are detected by VP test. Media and Reagent: MR-VP medium and Barritt’s Reagent A (contains alpha-naphthol) and Barritt’s Reagent B (contains KOH). Method: Inoculate medium and incubate at 37°C for 48 hours. After incubation, transfer 2.5 ml of inoculate to another tube and add six drops of Barritt’s Reagent A and two drops of Barritt’s Reagent B. Gently mix and let it sit for 10-15 minutes to allow time for color development. Expected results: Positive test: acetoin + alpha-naphthol + KOH = red color Negative test: alpha-naphthol +KOH = copper color

Results for VP test:

Results for VP test

Citrate Utilization:

Citrate Utilization Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coli Media and Reagent: Simmon’s Citrate Agar. It has citrase as the only carbon source and PH indicator bromothymol blue Method: Inoculate the slant and incubate at 37°C for 24-48 hours. Expected results: Positive test: Growth and color changes to blue Negative test: No growth and color remains green

Results for Citrate Test:

Results for Citrate Test Negative Positive

H2S Production:

H 2 S Production Bacteria use enzyme cysteine desulfurase to hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product. Media and Reagent: SIM with cysteine and ferrous sulfate (detects H2S) Method: Inoculate the media and incubate at 37°C for 24-48 hours. Expected Results: Positive Test: H2S production = Black Negative Test: No H2S production = No blackening of medium

Results of H2S production:

Results of H 2 S production Positive Negative

Urea Utilization:

Urea Utilization Some bacteria produce urease, an enzyme capable of breaking down urea and produce alkaline end products. This distinguishes Proteus from other bacteria Media and Reagent: Urea Broth with phenol red Method: Inoculate the media with a loop and incubate at 37°C for 24 hours. Expected Results: Positive test: production of alkaline end products = pinkish red color Negative test: No color change

Results for Urea Test:

Results for Urea Test Negative Positive

Motility Test:

Motility Test This is not a biochemical test, but it can distinguish bacteria. It determines presence of flagella. Media and reagent: Deep agar Method: Inoculate deep with a needle and incubate at 37°C for 24-48 hours. Expected results: Positive test: Growth spread away from the line of inoculation = motile Negative test: Growth only occurred at the line of inoculation = Non-motile

Results for Motility Test:

Results for Motility Test

Mannitol Fermentation:

Mannitol Fermentation Mannitol Salt Agar contains 7.5% NaCl , which is inhibitory to most bacteria. Bacteria that can grow on this agar can be differentiated based on mannitol fermentation. Fermentation of mannitol results in acidic products which turn phenol red pH indicator from red to yellow. Media and reagent: MSA and phenol red indicator Method: Streak MSA plate and incubate at 37°C for 2 days. Expected results: Positive test: Mannitol fermentation occurred = growth and color changed to yellow Negative test: No mannitol fermentation = may or may not grow and no color change

Results for mannitol fermentation:

Results for mannitol fermentation Positive Negative No Growth

Lactose Fermentation:

Lactose Fermentation MacConkey Agar contains bile salts and crystal violet, both inhibitory to Gram-positive bacteria and selects Gram-negative bacteria, such as E. Coli. It also differentiates lactose-fermenting bacteria, such as E. Coli from non-lactose fermenting bacteria. Media and Reagent: MacConkey Agar and neutral red dye Method: Streak MAC plate and incubate at 37°C for 2 days. Expected results: Positive test: Lactose fermentation = Growth and color change to pink Negative test: No lactose fermentation = May or may not grow and no color change

Results of Lactose Fermentation:

Results of Lactose Fermentation

Glucose Fermentation & Gas Production:

Glucose Fermentation & Gas Production How to Perform Test: Inoculate broth with inoculating loop. Property it tests for: This test is done to help differnetiate species of the family Enterobacteriaceae. This tests for the bacteria’s ability to ferment glucose and produce gas and/or an acid end-product.. Media and Reagents Used: Glucose broth contains beef extract, gelatine peptone, and glucose. A phenol red indicator is added to indicate an acid enproduct. A Durham tube is added to indicate gas production. Results A positive result for acid is yellow after indicator is added (indicating glucose fermentation) A positive result for gas is a bubble in the Durham tube. A completely negative result has no color change or reddish color and no bubble.

Sugar Fermentation Tests:

Sugar Fermentation Tests Tube 1: Negative acid /Negative gas Tube 2A: Must incubate longer (ambiguous result) Tube 2B: Positive acid /Negative gas Tube 3A: Positive acid/ Positive gas

Coagulase:

How to Perform Test: Inoculate rabbit plasma with one single colony. Break up colony and stir until blended in plasma. Incubate at 37 degrees C for 24 hours. Property it tests for: This tests for the bacteria’s ability to clot blood plasma using the enzyme coagulase. If the organism has coagulase it will clump rabbit plasma. Media and Reagents: This media contains rabbit plasma dissolved in buffer. Coagulase

Coagulase Results:

Coagulase Results Reading Results: If the organism is has coagulase it will clump the plasma. If the organism does not have coagulase it will not clump the plasma.

Thank You:

Thank You

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